Platelet transfusion is only one therapy against platelet depletion caused by e.g., bleeding associated with accidents and during use of anti-cancer agents. Platelet preparations to be used for the time are produced completely (100%) depending upon blood donation with good intentions, at present. Platelets are very fragile and a method enabling platelets for use in therapy to store for a long time has not yet been developed. Actually, it is reported that the storage life of platelets in the latest medical institutions is four days; however, in consideration of time required for inspection and shipment, substantial storage life thereof in clinical sites including clinics is conceivably about three days. Likewise, many blood banks have a difficulty in storing platelets while keeping freshness at all times. In addition, the supply amount of platelet preparations is likely to vary dependent upon a decrease of blood donors and an increase of blood donors affected with viral infectious diseases (non-patent documents 1, 2).
In the circumstances, recently, a novel platelet supply source has attracted attention, which has been developed in place of blood donation having such problems (non-patent document 3). As an example, development of a technique of producing a large amount of platelets in vitro using somatic stem cells, i.e., hematopoietic stem cells (umbilical cord blood stem cells) is known. However, this technique has not yet been put into practical use, because an in vitro method for proliferating hematopoietic stem cells per se has not yet been established. In contrast, pluripotent stem cells, i.e., embryonic stem (ES) cells have an advantage in that they can be unlimitedly proliferated in vitro and have attracted attention as a supply source for producing blood cells including platelets. In this respect, techniques for producing mature megakaryocytes and platelets from human ES cells have been already reported (non-patent documents 4, 5). However, in the techniques (methods), the production efficiency of platelets is low and tens of thousands of petri dishes are required for producing a single blood transfusion preparation. These methods were insufficient from a practical point of view.
In transfusion of platelets, refractory to platelet transfusion is raised as a problem. At the first-time transfusion, platelets having a different human leukocyte antigen (HLA) from a patient can be used; however, a specific antibody against the HLA is produced in the patient's body when transfusion is repeated, with the result that the platelets are rejected immediately upon transfusion. In addition, since platelets have own blood type, i.e., an allogeneic human platelet antigen (HPA), refractory to transfusion caused by incompatibility of HPA types is also known. As a technique which can overcome this problem, techniques for producing megakaryocytes and platelets from human induced pluripotent stem (iPS) cells have been reported (non-patent documents 6, 7). For example, if platelets are induced from a patient-derived iPS cells, it is theoretically possible to produce a rejection-free custom-made platelet preparation. However, in producing platelets from iPS cells, at least about 50 days are required for producing platelets from fibroblasts (non-patent documents 6, 7). For the reason, this production method was insufficient from a practical point of view. In the meantime, as a method for producing platelets from fibroblasts, a technique called direct reprogramming is known (non-patent document 8). According to this technique, the period required for producing platelets will be greatly shorter than the method for producing platelets via iPS cells. Advantageously, platelets are produced in about 14 days. However, the direct reprogramming using fibroblasts requires gene introduction. The effect of the presence of a gene transfer vector on safety is concerned.
As a culture medium for inducing differentiation of hematopoietic stem cells into megakaryocytes/platelets, MKLI medium (megakaryocyte lineage induction medium) is known. The MKLI medium is a medium prepared by adding, 2 mM L-glutamine, a 100 U/mL penicillin-streptomycin solution, 0.5% bovine serum albumin, 4 μg/mL LDL cholesterol, 200 μg/mL iron-saturated transferrin (iron-bound transferrin), 10 μg/mL insulin, 50 μM 2-β-mercaptoethanol, nucleotides (20 μM for each of ATP, UTP, GTP and CTP) and 50 ng/mL thrombopoietin (thrombopoietin: TPO) to Iscove's Modified Dulbecco's Medium (IMDM) (non-patent document 9). The present inventors have so far conducted studies on a technique for inducing differentiation of cells excluding hematopoietic stem cells into megakaryocytes/platelets. As a result, they have found that if preadipocytes derived from a human subcutaneous adipose tissue (non-patent documents 9, 10) and mouse-derived preadipocytes (non-patent documents 9, 11) are cultured in the MKLI medium, they can be differentiated into megakaryocytes/platelets. In these methods using preadipocytes, platelets can be efficiently produced in vitro in a relatively short period of time. In addition, these methods do not require gene introduction and they are excellent in safety when the platelets are administered to patients. In the context, a method for preparing megakaryocytes and platelets at lower cost or more efficiently has been desired.
“Platelets” is one of material components in blood and plays a major role in arresting bleeding in living bodies. If abnormal clump of platelets is formed, thrombotic diseases are caused. Platelets are also involved in metastasis and growth of cancer. The role of platelets has recently attracted attention in a wide variety of fields. Platelets are developed from hematopoietic stem cells in the bone marrow through the following step. The hematopoietic stem cells are developed into megakaryocyte lineage progenitor cells and then into megakaryoblasts, which are further matured into megakaryocytes. Thereafter, the cytoplasm of megakaryocytes is torn apart into several thousands of pieces and released into blood. It has been considered that, in order to form megakaryocyte colonies from hematopoietic stem cells in the bone marrow, two types of factors having different actions are required (non-patent document 12), more specifically, Meg-CSF, which supports colony formation by itself, and Meg-POT, which does not functionally support formation of colonies but promotes maturation of megakaryocytes in the presence of Meg-CSF. As the factor having Meg-CSF activity in human, e.g., IL-3 (non-patent document 13) and GM-CSF (non-patent document 14), are known. As the factor having Meg-POT activity in human, e.g., IL-6 (non-patent document 15), IL-11 (non-patent document 16) and LIF (non-patent document 17) are known.
However, these are all not specific factors to the megakaryocyte/platelet lineage but factors known to act on other hematocyte system and cells other than hematocytes. Thus, if these are administered as pharmaceutical products in expectation of action on the megakaryocyte/platelet lineage, it is concerned that another action is expressed against expectation. In the context, a physiologically active substance specifically acting on megakaryocyte/platelet lineage and extremely useful as a pharmaceutical product has been strongly desired. As a factor specifically acting on the megakaryocyte/platelet lineage, a human c-MPL receptor ligand, TPO, is known and a gene of TPO has been cloned (non-patent document 18). The c-MPL protein is a glycoprotein, which expresses on hematopoietic stem cells and megakaryocyte lineage cells and belongs to a cytokine receptor gene family. It has been suggested that the c-MPL protein is deeply involved in platelet production as a receptor for a novel factor involved in platelet production. TPO cloned has both Meg-CSF activity and Meg-POT activity and serves as a specific factor to the megakaryocyte/platelet lineage.
TPO is an important regulatory factor of platelet production and stimulates growth of megakaryocytes producing platelets and production of platelets from megakaryocytes (non-patent document 19). TPO is synthesized in the liver as a proprotein consisting of 353 amino acids and becomes a mature protein molecule by cleaving a signal peptide of 21 amino acids. The mature protein molecule consists of two domains having a high homology with erythropoietin and a highly glycosylated carboxy terminal important for protein stability (non-patent document 20). An increase of TPO level is not observed in patients with immune thrombocytopenic purpura (ITP) (non-patent document 21). The need for mass production and purification of TPO has been insisted for developing a drug for increasing platelets; however, since TPO is consistently produced from major TPO-producing cells, i.e., hepatocytes, in an extremely low amount, supplying purified TPO endogenously produced in a large amount has not been achieved.
In the context, studies on recombinant TPO have been conducted and two types of recombinant TPO molecules have been subjected in a large-scale clinical trial. One is recombinant human TPO (referred to also as either rHuTPO or rHTPO), which is a glycosylated molecule having the same full-length amino acid sequence as in natural TPO. The other one is a non-glycosylated molecule containing 1-163 amino acids corresponding to a biologically active domain of natural TPO, namely a polyethylene glycol (PEG)-bound recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) (non-patent documents 19, 20, 22). Both recombinant TPO molecules are potent stimulatory substances for platelet production in human and have an ability to mitigate thrombocytopenia caused by chemical therapy and are useful for reducing necessity of platelet transfusion (non-patent documents 23, 24). In the context, an attempt to express a full length recombinant human TPO (rHuTPO) has been made using cultured cells; however, glycosylation of the resultant TPO was different from endogenous TPO. Thus, a technology for producing TPO having an ability to induce/promote platelet production by such a method has not yet been established. It was also found in clinical researches conducted over the past decade that PEG-rHuMDGF induces an antibody that cross-reacts with endogenous TPO, and induces thrombocytopenia in 4% of healthy individuals and 0.6% of cancer patients who received intensive chemotherapy (non-patent document 22).
In such technological circumstances, practical technologies for TPO production, such as TPO production using hepatocytes and recombinant TPO production without the aforementioned problems, have been desired.
As a medium capable of inducing differentiation of hematopoietic stem cells into megakaryocytes/platelets, MKLI medium (megakaryocyte lineage induction medium) is known. The MKLI medium is a medium prepared by adding 2 mM L-glutamine, a 100 U/mL penicillin-streptomycin solution, 0.5% bovine serum albumin, 4 μg/mL LDL cholesterol, 200 μg/mL iron-saturated transferrin (iron-bound transferrin), 10 μg/mL insulin, 50 μM 2-β-mercaptoethanol, nucleotides (20 μM each for ATP, UTP, each GTP and CTP) and 50 ng/mL TPO to Iscove's Modified Dulbecco's Medium (IMDM) (non-patent document 9). The present inventors have so far conducted studies on a technology for inducing differentiation of cells excluding hematopoietic stem cells into megakaryocytes/platelets, and have found that if human preadipocytes derived from a subcutaneous adipose tissue (non-patent documents 9, 10) and mouse-derived preadipocytes (non-patent documents 9, 11) are cultured in the MKLI medium, these preadipocytes can be differentiated into megakaryocytes/platelets. However, mesenchymal cells such as preadipocytes and mesenchymal cell-derived megakaryocytes produce TPO having a differentiation-inducing property to platelets have not yet been reported.
It has been also known that if preadipocytes are cultured in a culture medium containing dexamethasone, 3-isobutyl-1-methylxanthine, insulin and indomethacin, they are induced to differentiate into adipose cells (non-patent document 25). However, it has been believed that TPO is produced in the hepatocytes; and never known that TPO is produced during the process of differentiating preadipocytes into adipose cells.
Note that, in connection with cell-surface markers to be used in the present invention, the following facts are known. CD31 is expressed on e.g., vascular endothelial cells and involved in adhesion between cells. c-MPL protein (Myeloproliferative leukemia protein) is a receptor for thrombopoietin having differentiation/growth action of megakaryocytes producing platelets. c-MPL is expressed not only on platelets and megakaryocytes but also on erythroblasts. CD71 is a type II membrane glycoprotein and known as a transferrin receptor. CD71 is expressed not only on activated T cells and activated B cells but also on macrophages and all proliferating cells.